The human IgH locus contains nine functional C genes and two pseudogenes, arranged 5′ Cμ-Cδ-Cγ3-Cγ1-ψCε-Cα1-ψCγ-Cγ2-Cγ4-Cε-Cα2 3′, over a 350 kb region of chromosome 14 (1). Overlapping phage and cosmid clones established the C gene organisation but attempts to obtain the entire region on overlapping clones or a single yeast artificial chromosome (YAC)5 have been unsuccessful. PCR-based approaches identified highly repetitive regions downstream of Cα1 and Cα2 which include virtually identical 3′ enhancers made up from different numbers of short motifs (3, 4). Similar repetitiveness, leading to instability, was also assumed for the estimated 40–70 kb gap between Cδ and Cγ3, which could not be cloned to establish a C gene contig. Indirect results from transgenic mice lacking different regions 3′ of Cμ and Cδ further suggested that this particular downstream region might be important for high expression and switching of IgH genes (5, 6). Analysis of recombination in the Cδ-Cγ3 interval showed a lack of association between these genes, which may indicate a potential hot spot for recombination (7, 8). The potential significance of this region is further supported by the finding that a large area between Cδ and Cγ3 is deleted in certain leukemias, which may be linked to a pathogenic mechanism active at an early stage of B-cell development (9).
In the mouse, the IgH locus has been completely cloned (10) and in DNA-binding assays a cluster of matrix association regions (MARs) was found in the Cδ-Cγ3 intron (11). Although the region was not extensively characterized by sequencing, the presence of long interspersed repetitive elements in the vicinity of MARs may lead to the high recombination observed in this region of the IgH locus. Probes derived from bacteriophage clones covering the mouse Cδ-Cγ3 region failed to identify corresponding sequences in the human locus (9).
During B lymphocyte development it is generally thought that transcriptional activation of the IgH locus is regulated using two enhancer arrays which flank the constant region cluster (12–14, reviewed in 15). These arrays, the Eμ intron enhancer and the 3′ enhancer downstream of Cα, contain multiple sites for the binding of both tissue specific and ubiquitous trans-acting factors (13, 16). Enhancer-mediated activation appears to be controlled by the interaction of both negative and positive regulatory elements (17, 18). The Eμ intron enhancer provides potential protein binding sites for several regulatory elements which are essential for lymphocyte differentiation, including E47, PU.1, Ets-1, TFE3, USF and Oct (19, 20). The 3′α enhancer shares some DNA sequence elements with the Eμ enhancer but also has additional motifs for factors involved in transcriptional regulation (13, 16).
Activation and sequential DNA rearrangement of the Ig loci are crucial steps in antibody expression and cis-acting locus elements like enhancers, which accommodate various combinations of factor-binding sites, have been implicated in IgH locus recombination and transcription (17). Although important information about enhancer core functions has been obtained from mutant mouse strains, these are poorly understood processes as there appears to be no activity of either enhancer in early B-cell development when IgH heavy chain rearrangement is initiated. For example, deletion of the heavy chain intron enhancer Eμ showed severe impairment of VH to DJH rearrangement whilst the earlier D to JH rearrangement was much less affected (21). In chimeric mice, in which the Cα3′ enhancer was replaced by a marker gene, isotype deficiency and impairment of heavy chain class-switching were observed (18).